LOCUS       sequence-261481-        7404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knock in H2B-GFP into the human PAX6 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7404)
  AUTHORS   Tchieu J, Zimmer B, Fattahi F, Amin S, Zeltner N, Chen S, Studer L
  TITLE     A Modular Platform for Differentiation of Human PSCs into All Major 
            Ectodermal Lineages.
  JOURNAL   Cell Stem Cell. 2017 Sep 7;21(3):399-410.e7. doi: 
            10.1016/j.stem.2017.08.015.
  PUBMED    28886367
REFERENCE   2  (bases 1 to 7404)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stem.2017.08"; journalName: "Cell Stem Cell"; date: 
            "2017-09-7- 7"; volume: "21"; issue: "3"; pages: "399-410.e7"
COMMENT     Sequence Label: PAX6 TALEN L
FEATURES             Location/Qualifiers
     source          1..7404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      649..658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             658..723
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             730..750
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2356..2943
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     primer_bind     complement(2961..2978)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2967..3078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3196..3216)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3201..3530
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3381..3516
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3443..3462
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3579..4604
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3579..4604
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4734..4855
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4771..4790)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4825..4844
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4908..5699,5700..5768))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5531..5550
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5769..5860)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5902..5921
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5927..5946
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6032..6620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6521..6540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6774..6791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6889..6906
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6890..6906
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7362..7378
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctataggggc caccatggac
      661 tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat
      721 aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgtagat
      781 ttgagaactt tgggatattc acagcagcag caggaaaaga tcaagcccaa agtgaggtcg
      841 acagtcgcgc agcatcacga agcgctggtg ggtcatgggt ttacacatgc ccacatcgta
      901 gccttgtcgc agcaccctgc agcccttggc acggtcgccg tcaagtacca ggacatgatt
      961 gcggcgttgc cggaagccac acatgaggcg atcgtcggtg tggggaaaca gtggagcgga
     1021 gcccgagcgc ttgaggccct gttgacggtc gcgggagagc tgagagggcc tccccttcag
     1081 ctggacacgg gccagttgct gaagatcgcg aagcggggag gagtcacggc ggtcgaggcg
     1141 gtgcacgcgt ggcgcaatgc gctcacggga gcacccctca acctgacccc agagcaggtc
     1201 gtggcaattg cgagccatga cgggggaaag caggcactcg aaaccgtcca gaggttgctg
     1261 cctgtgctgt gccaagcgca cggacttacg ccagagcagg tcgtggcaat tgcgagccat
     1321 gacgggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg
     1381 cacggactga ccccagagca ggtcgtggca attgcgagca acatcggggg aaagcaggca
     1441 ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacggact tacacccgaa
     1501 caagtcgtgg caattgcgag caacggaggg ggaaagcagg cactcgaaac cgtccagagg
     1561 ttgctgcctg tgctgtgcca agcgcacgga cttacgccag agcaggtcgt ggcaattgcg
     1621 agcaacatcg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc
     1681 caagcgcacg gactaacccc agagcaggtc gtggcaattg cgagccatga cgggggaaag
     1741 caggcactcg aaaccgtcca gaggttgctg cctgtgctgt gccaagcgca cgggttgacc
     1801 ccagagcagg tcgtggcaat tgcgagcaac ggagggggaa agcaggcact cgaaaccgtc
     1861 cagaggttgc tgcctgtgct gtgccaagcg cacggcctga ccccagagca ggtcgtggca
     1921 attgcgagca acaacggggg aaagcaggca ctcgaaaccg tccagaggtt gctgcctgtg
     1981 ctgtgccaag cgcacggact gacaccagag caggtcgtgg caattgcgag caacaacggg
     2041 ggaaagcagg cactcgaaac cgtccagagg ttgctgcctg tgctgtgcca agcgcacgga
     2101 ctcacgcctg agcaggtagt ggctattgca tccaacaacg ggggcagacc cgcactggag
     2161 tcaatcgtgg cccagctttc gaggccggac cccgcgctgg ccgcactcac taatgatcat
     2221 cttgtagcgc tggcctgcct cggcggacga cccgccttgg atgcggtgaa gaaggggctc
     2281 ccgcacgcgc ctgcattgat taagcggacc aacagaagga ttcccgagag gacatcacat
     2341 cgagtggcag gttcccaact cgtgaagagt gaacttgagg agaaaaagtc ggagctgcgg
     2401 cacaaattga aatacgtacc gcatgaatac atcgaactta tcgaaattgc taggaactcg
     2461 actcaagaca gaatccttga gatgaaggta atggagttct ttatgaaggt ttatggatac
     2521 cgagggaagc atctcggtgg atcacgaaaa cccgacggag caatctatac ggtggggagc
     2581 ccgattgatt acggagtgat cgtcgacacg aaagcctaca gcggtgggta caatcttccc
     2641 atcgggcagg cagatgagat gcaacgttat gtcgaagaaa atcagaccag gaacaaacac
     2701 atcaatccaa atgagtggtg gaaagtgtat ccttcatcag tgaccgagtt taagtttttg
     2761 tttgtctctg ggcatttcaa aggcaactat aaggcccagc tcacacggtt gaatcacatt
     2821 acgaactgca atggtgcggt tttgtccgta gaggaactgc tcattggtgg agaaatgatc
     2881 aaagcgggaa ctctgacact ggaagaagtc agacgcaagt ttaacaatgg cgagatcaat
     2941 ttccgctcat aaaaaatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3001 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3061 aaatgaggaa attgcatcac aacactcaac cctatctcgg tctattcttt tgatttataa
     3121 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3181 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3241 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3301 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3361 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3421 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3481 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3541 gagcttgtat atccattttc ggatctgatc agcacgtgat gaaaaagcct gaactcaccg
     3601 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     3661 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     3721 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     3781 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     3841 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     3901 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     3961 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     4021 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     4081 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     4141 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     4201 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     4261 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     4321 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     4381 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     4441 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     4501 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     4561 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgtg ctacgagatt
     4621 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4681 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4741 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4801 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4861 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta ccaatgctta
     4921 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4981 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag cgctgcgatg
     5041 ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca gccagccgga
     5101 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5161 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatc
     5221 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5281 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5341 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5401 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5461 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5521 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5581 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5641 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5701 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5761 atactcatat tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5821 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggtcag tgttacaacc
     5881 aattaaccaa ttctgaacat tatcgcgagc ccatttatac ctgaatatgg ctcataacac
     5941 cccttgctca tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt
     6001 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6061 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6121 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6181 ttctagtgta gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc
     6241 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6301 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6361 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6421 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6481 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6541 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6601 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6661 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6721 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6781 cagtgagcga ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa
     6841 ggcccctgac ggatggcctt tttgcgtttc tacaaactct ttctgtgttg taaaacgacg
     6901 gccagtctta agctcgggcc ccctgggcgg ttctgataac gagtaatcgt taatccgcaa
     6961 ataacgtaaa aacccgcttc ggcgggtttt tttatggggg gagtttaggg aaagagcatt
     7021 tgtcagaata tttaagggcg cctgtcactt tgcttgatat atgagaatta tttaacctta
     7081 taaatgagaa aaaagcaacg cactttaaat aagatacgtt gctttttcga ttgatgaaca
     7141 cctataatta aactattcat ctattattta tgattttttg tatatacaat atttctagtt
     7201 tgttaaagag aattaagaaa ataaatctcg aaaataataa agggaaaatc agtttttgat
     7261 atcaaaatta tacatgtcaa cgataataca aaatataata caaactataa gatgttatca
     7321 gtatttatta tcatttagaa taaattttgt gtcgccctta attgtgagcg gataacaatt
     7381 acgagcttca tgcacagtgg cgtt
//
